The overall goal of the proposed research program is to define the mechanism by which virion glycoproteins of HSV mediate virus entry into neuronal and non-neuronal cells. After the initial interaction virion gC and host cell heparin sulfate, four glycoproteins, gB, gD and gH/gL complex mediate virus-cell fusion at the plasma membrane. A major function gD is to interact with a specific cellular receptor which apparently triggers later events involving gB and gH/gL. A recent finding from Dr. Patricia Spear's laboratory (Northwestern University has shown that a protein designated HVEM (herpes virus entry mediator), when expressed by normally non-permissive cells, makes those cells permissive for HSV-1 entry. HVEM is a member of the tumor necrosis factor receptor protein family. The co-principal investigators, Drs. Eisenberg and Cohen, have initiated a collaboration with Dr. Spear to study the interaction of gD and HVEM. Drs. Eisenberg and Cohen have subcloned a truncated form of the HVEM gene (HVEMt) into the baculovirus expression system and found that purified truncated gD (gDt) binds directly to purified HVEMt in vitro. Thus, the two proteins can interact directly, without any requirement for another viral or cellular protein. They propose to extend these initial observations and to further detail the stoichiometry and affinity of this interaction, as well as to attempt co-crystallization. gD and HVEM mutants will be used to localize the binding sites and to measure the effect of mutation on the affinity of the interaction. They will explore how the interaction between gD and HVEM triggers the next steps of virus entry involving other viral and/or possibly other cellular molecules. This will be done both in vitro with purified glycoproteins, and in vivo, with virions and cells. Preliminary data suggest that mutations in gD can either enhance or ablate binding to HVEM. It is possible that gD interacts with more than one receptor, depending on the cell type and differences in gD structure. They will explore whether strain differences in gD affect viral tropism, especially in viral isolates from encephalitis patients versus isolates from other sites in the human host. Three specific aims are proposed: (1) to characterize the interaction between gD and HVEM in vitro; (2) to localize domains involved in the GD-HVEM interaction; and (3) to examine the consequences of gD-receptor interactions for virus entry into neuronal and non-neuronal cells.